Semi-enzymatic synthesis and application of 13C-isotopically labelled inositol-(1,4,5)-trisphosphate†

Abstract

Inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P₃) is a crucial secondary messenger that controls calcium (Ca²⁺) levels inside cells, yet many questions regarding Ins(1,4,5)P₃ metabolism are challenging to address with current methods. Here, a semi-enzymatic milligram scale synthesis of isotopically labeled [¹³C₆]Ins(1,4,5)P₃ is reported which then served as a substrate to monitor the activity of mammalian type II inositol 1,4,5-trisphosphate 5-phosphatase INPP5B, using NMR spectroscopy in real time. In addition, the phosphorylation sequence catalyzed by inositol polyphosphate multikinase IPMK was confirmed using [¹³C₆]Ins(1,4,5)P₃ and 2D NMR spectroscopy. The method was subsequently applied to characterize the phosphorylation/dephosphorylation reactions of a putative inositol phosphate kinase from the alga Klebsormidium nitens (KnIPK2). KnIPK2 displayed 6-kinase activity towards [¹³C₆]Ins(1,4,5)P₃, and dual 4/6- and 5-phosphatase activity towards [¹³C₆]Ins(1,3,4,5,6)P₅. Finally, [¹³C₆]Ins(1,4,5)P₃ was utilized as an internal standard in hydrophilic liquid interaction chromatography mass spectrometry (HILIC-MS) experiments, to quantify dephosphorylation of Ins(1,4,5)P₃ by INPP5B. [¹³C₆]Ins(1,4,5)P₃ therefore constitutes a broadly applicable analytical tool that should facilitate the characterization of Ins(1,4,5)P₃ metabolism in the future.